The objective of our research is elucidation of the molecular mechanism of a variety of heme and non-heme proteins both isolated and in intact cells using the techniques of magnetic resonance spectroscopy. It is our belief that an accurate knowledge of the mechanisms of protein function is fundamental to an understanding of the molecular basis of life and may be essential for dealing with health related problems arising from molecular abnormalities. The proteins chosen for this investigation are: human hemoglobins, phosphoryl transfer enzymes (kinases and synthetases), cytochrome c, carbonic anhydrase, antitumor antibiotics (neocarzinostatin and bleomycin) and insulin. For in vivo studies of protein function, human normal and sickle red blood cells, lymphocytes and frog muscles will be used. With human hemoglobin, the mode of oxygen binding, the binding of organophosphates and the mechanism of cooperative oxygenation, will be studied. Comparative 31p NMR studies of human normal and sickle blood, in the presence of varying levels of intracellular 2,3-DPG, may shed light on the nature of intracellular aggregation of sickle hemoglobin. The arrangement and conformation of substrates at the catalytic sites of several kinases and synthetases and the role of metal ions in the catalysis of phosphoryl transfer will be studied by NMR methods. Arrangement of substrates and products at the active site of carbonic anhydrase will be studied by nuclear relaxation techniques. The electron transfer mechanism of cytochrome c and its ion-binding properties will be elucidated by determing the relative positions of the metal centers in the cytochrome c- cytochrome oxidase complex and by locating the site for chromous ion reduction of cytochrome c using nuclear relaxation techniques and Cr3 ion as the paramagnetic reference point. The existence and conformation of a Fe-bleomycin-DNA ternary complex and the conformation and mechanism of neocarzinostatin will be studied by 1H and 13C NMR spectroscopy. The effect of insulin on intracellular pH will be studied by 31P NMR of intact muscle. A secondary objective of our research is the development of new methodology and/or refinement of existing NMR methods for the study of biological systems.